Low-dose of Acetylsalicylic Acid Upregulates Expression of eNOS

mRNA and Downregulates Interleukin-6 (IL-6) and Transforming

Growth Gactor-β1 (TGF-β1) mRNA in Rat Kidney of Preeclampsia

Model

Yuyun Nailufar

, Rul Afiyah Syarif

, Andi Fitriani Kusuma

, Farmita Chairani

Nia Marlina

, Charolina Vivi Vienetta

, M. Hadri Ar-Ridho

, Totok Utoro

, Nur Arfian

Department of Anatomy, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta,

Indonesia

Department of Pharmacology and Therapy, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada,

Yogyakarta, Indonesia

Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia

Department of Anatomical Pathology, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada,

Yogyakarta, Indonesia

Keywords: Preeclampsia, Inflammation, Kidney Injury, IL-6, TGF-β1, Endothelial Dysfunction

Abstract: Preeclampsia leads to endothelial dysfunction and kidney injury with inflammation and fibrosis. Low-dose

acetylsalicylic acid (ASA) administration may decrease uterine artery resistance; however, its effect on the

kidney has not been elucidated yet. We performed a preeclampsia model in pregnant female Wistar rats (PE

group, n=5, 150-200 grams) using L-NAME 50mg/kg of BW intraperitoneal injection on day 1-18 of

pregnancy. Low doses of ASA with dose 75 (PE+ASA75) and 125 (PE+ASA125) mg/kg body weight were

administered in preeclampsia rats in the day 10-12 day of pregnancy. The Control group was normal pregnant

rats with NaCl treatment (n=5). On day 18, rats were sacrificed; kidneys were harvested, then extracted for

Reverse Transcriptase-PCR (RT-PCR) of eNOS, TGF-β1, and IL-6 mRNA expression measurements.

Proteinuria and rat blood pressure were measured before termination. L-NAME injection-induced

preeclampsia showed significantly higher systolic blood pressure and proteinuria in the PE group than in the

control group (p<0.05). However, there were no changes in podocin and nephrin expression. In conclusion,

the low dose of ASA 125mg/Kg BW ameliorates kidney inflammation and TGF-β1 expression in the rat

preeclampsia model's kidney.

1 INTRODUCTION

The mortality of pregnant women still becomes a

health problem, especially in developing countries.

https://orcid.org/0000-0001-8281-4769

https://orcid.org/0000-0001-8114-9322

https://orcid.org/0000-0002-4803-1434

https://orcid.org/0000-0003-1515-7003

https://orcid.org/0000-0002-4041-8087

https://orcid.org/0000-0001-6082-4448

https://orcid.org/0000-0002-9543-3110

https://orcid.org/0000-0002-1823-6295

https://orcid.org/0000-0003-1694-2054

The major causes of maternal mortality during 2010-

2013 are bleeding, hypertension during pregnancy,

and abortion (Linggardini and Aprilina, 2016). In

recent years preeclampsia and eclampsia are the

leading causes of maternal mortality (Pellicer et al.,

Nailufar, Y., Syarif, R., Kusuma, A., Chairani, F., Marlina, N., Vienetta, C., Ar-Ridho, M., Utoro, T. and Arﬁan, N.

Low-dose of Acetylsalicylic Acid Upregulates Expression of eNOS mRNA and Downregulates Interleukin-6 (IL-6) and Transforming Growth Gactor-1 (TGF-1) mRNA in Rat Kidney of

Preeclampsia Model.

DOI: 10.5220/0010487800630068

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 63-68

ISBN: 978-989-758-499-2

2011), with the number of preeclampsia in

developing countries ranging from 2-8%.

Preeclampsia is a condition in pregnancy

characterized by hypertension (systolic pressure ≥140

mmHg and diastole ≥90 mmHg) and proteinuria ≥300

mg in24 hours. There is still no effective treatment for

preeclampsia (Kemenkes RI, 2014; Rachmi and

Sulistyono, 2016).

The pathogenesis of preeclampsia is related to

trophoblast invasion of the spiral arteries of the

uterus. This condition causes decreased

uteroplacental blood flow and placental ischemia.

Placental ischemia triggers tissue hypoxia, a release

of oxidative stress, and anti-angiogenic factors'

release into the maternal circulation, such as sFlt1.

Increased levels of sFlt1 can cause a decrease in

vascular endothelial growth factor (VEGF) and

placental growth factor (PIGF) levels. VEGF and

PIGF play a role in maintaining the integrity of

endothelial cells in the body. Decreased VEGF and

PIGF in preeclampsia are believed to be the cause of

systemic endothelial cell dysfunction and

microangiopathy (Oparil et al., 2003).

One genetic predisposition that plays a role in

triggering preeclampsia is an endothelial nitric oxide

synthase (eNOS) gene disorder regulating nitric oxide

activation. Nitric oxide (NO) is an endothelial

vasodilator with functions as antithrombotic and

atheroprotective. Under normal circumstances, the

NO pathway is activated and increases the levels of

NO in vessels. Increased NO levels are responsible

for the maternal vasodilatation required to

accommodate increased circulating volume during

pregnancy without raising blood pressure. However,

in preeclampsia, this process of adaptation is

disrupted, an endothelial disorder occurs. Thus blood

pressure increases, and proteinuria develops. The

decrease in NO production is associated with

polymorphisms in genes that regulate NO production,

namely the endothelial nitric oxide synthase (eNOS)

gene (Suharto et al., 2014).

Endothelial impairment in the kidneys caused by

a decrease in VEGF will lead to glomerular capillary

endothelins and proteinuria. Several studies have

shown that interleukin-6 (IL-6) and transforming

growth factor-β1 (TGF-β1) play a role in renal

disease progression. IL-6 can cause kidney disease by

increasing the tubular epithelial cell signal response

to a pro-fibrotic growth factor such as TGF-β1.

Increased expression of TGF-β1 mRNA may induce

renal fibrosis by producing extracellular matrix

(ECM). Fibrosis can lead to more severe

tubulointerstitial damage resulting in a progressive

decline in the nephron and renal function. The IL-6

and TGF-β1 role, both acutely and chronically in

renal tubular damage in the preeclampsia model, has

not been widely known (Munkhaugen and Vikse,

2009; Jones et al., 2015).

One of the therapeutic management in the case of

preeclampsia is the administration of acetylsalicylic

acid. Low-acetyl-salicylic acid administration may

improve blood vessel circulation and prevent

vasoconstriction, resulting in increased organ

perfusion and preventable kidney damage. Acetyl-

salicylic acid can also stimulate the activation of

eNOS that catalyzes NO synthesis for hypertensive

patients in preeclampsia (Danuyanti et al., 2018).

Therefore, we need to observe the effect of low dose

acetylsalicylic acid administration on renal function

through eNOS, IL-6, TGF-β expression of mRNA on

the preeclampsia model.

2 MATERIALS AND METHODS

2.1 Preparation of Experimental

Animal

This research used Wistar strain female rat obtained

from Integrated Research and Testing Laboratory

(LPPT) Universitas Gadjah Mada with number

00104/04/LPPT/VIII/2017. Wistar strain female rats

with age ± 12 weeks, and bodyweight 150-200 grams

were used for the experiment. The rats were divided

into 4 groups: Control group (normotensive pregnant

rat), PE group (preeclamptic model rat induced with

L-NAME of doses 50 mg/kg BW/day from the first

day to 18th day of pregnancy), PE+ASA75 group

(preeclamptic model and treatment of acetyl-

salicylate acid of doses at 75 mg/Kg BW from the

tenth day until the twelfth day of pregnancy), and

PE+ASA125 group (preeclamptic model rad and

treatment of acetyl-salicylate acid of doses at 125

mg/Kg BW). The doses of ASA was determined by

rat bodyweight that was quantified daily.

2.2 Process of Impregnating Rat

The rats' conception was performed at the Faculty of

Pharmacy Laboratory Unit V by placing one male rat

and two female rats in one cage. The experimental

animals were mixed in the afternoon at around 4 pm

and then check for the vaginal plugs (copulation plug)

on the following morning at 6 am. The presence of

vaginal plugs (copulation plug) in animals was

calculated as the 1st day of pregnancy (Han et al.,

2015; Kaya et al., 2011).

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

2.3 Process of Measuring Blood

Pressure, Proteinuria, and

Preeclampsia Induction

Blood pressure measurement of an experimental

animal was performed using a non-invasive

sphygmomanometer. Blood pressure was measured

five times during the study: a day before mating Day-

0 (D-0), the 5th day of pregnancy (D-5), D-8, D-11,

D-13, and before surgery (D-18). Examination of

proteinuria using uriscan 3 GPH strips. Proteinuria

was measured on the day before mating (D-0), the 6th

day of pregnancy (D-6), D-9, D-12, D-14, and D-19.

Preeclampsia induction was done by dissolving L-

NAME 50 mg/kg BW/day with the gastric probe on

the first day of pregnancy until the 18th day of

pregnancy (Szalai et al., 2015). Each rat weighing

150-200 g was given L-NAME of 1.5 to 2 mL per rat.

2.4 Acetyl-salicylate Acid Therapy and

Proteinuria Examination

Acetyl-salicylate acid therapy was performed by

dissolving 75 mg and 125 mg tablets using aquadest

first. Acetyl-salicylate acid was given orally with the

probe to mice at 10 to 12 days of gestational age. Each

preeclampsia mice with a weight range of 150-200

grams got acetyl-salicylate acid therapy that has been

diluted as much as 1.35 to 1,8 mL/day in 10 to 12 days

of gestational age. Proteinuria examinations using

uriscan 3 GPH strips were performed at: days before

mating (D-0), the 5th day of pregnancy (D-5), D-8,

D-11, D-13 D-18.

2.5 Kidney Harvesting

After being treated for eighteen days, all groups were

terminated 24 hours after the treatment. After

anesthetized using ketamine (dose 60-100 mg/kg

BW), rats were sacrificed with a lethal dose of

ketamine. Rat's abdomen and thorax were opened,

kidneys were harvested. The left kidney was kept in

RNA later® for RNA extraction, and the right kidney

was fixated in 4% PFA in PBS for paraffin making.

2.6 RNA Extraction, cDNA Making,

and Reverse Transcription-PCR

(RT-PCR)

Examination of eNOS, TGF-β1, IL-6, and GAPDH

mRNA expression was done using RT-PCR

(Transcription Reaction of Polymerase Chain

Reactions). Kidney tissue was extracted using Trizol

RNA solution (GENEzolTM; Cat. No. GZR100).

RNA concentrations were quantified using nanodrop.

The cDNA was synthesized using 5xRT-buffer

(Toyobo, TRT-101), random primer (TAKARA®,

3801), dNTP (TAKARA®, 4030), ReverTra-Ace

(Toyobo®; TRT-101).Reverse transcriptase-

polymerase chain reaction (RT-PCR) was carried out

to examine the following cDNAs: eNOS 5 ’ -

GTCCTGCAAACCGTGCAGAG-3’(forward) and

5- TGGGTGCGCAATGTGAGTC-3 ’ (reverse);

TGF- β 1 5-CCGTGGCTTC TAGTGCTGAC-3'

(forward), and 5'-GGCGTTGTTGCG TTAGATAC-

3' (reverse); IL-6 5-GCCCTTCAGGA

ACAGCTATGA-3 ′(forward) and 5 ′-TGTCAA

CAACATCAGTCCCAAAGA-3 ′ (reverse); and

GAPDH 5’-CCCCCAATGTATCCGTTGTG-3’

(forward) and 5'TAGCCCAGGATGCCCTTGAGT-

3'(reverse). Then a 35 cycle PCR was carried out with

a denaturation condition of 94° C for 10 seconds,

annealing at 60° C for 30 seconds and an extension of

72° C for the 1-minute final extension phase ending

with a 72° C condition for 10 minutes.

2.7 Histopathological Examination

The kidney paraffin blocks were cut in 4 μm thickness

for Periodic Acid Schiff (PAS) staining to assess

glomerulosclerosis. The preparation was examined

under a light microscope (Olympus CX22®), an

image was captured using OptiLab software in 400×

magnification with a random area.

2.8 Statistical Analysis

Data were analyzed using a one-way ANOVA test for

normally distributed data and Kruskal-Wallis for data

that were not normally distributed. The value of

p<0.05 was considered statistically significant.

Statistical analyses were accomplished using SPSS

Software version 23 (SPSS Inc., Chicago).

2.9 PE Condition after L-NAME

Treatment

L-NAME treatment in pregnant rats induced PE

conditions as shown by higher proteinuria scores and

higher systolic blood pressure in the PE group than

the control group (Fig.1). However, both ASA treated

groups did not demonstrate attenuation of blood

pressure and proteinuria score.

Low-dose of Acetylsalicylic Acid Upregulates Expression of eNOS mRNA and Downregulates Interleukin-6 (IL-6) and Transforming

Growth Gactor-1 (TGF-1) mRNA in Rat Kidney of Preeclampsia Model

Figure 1: L-Name treatment demonstrated PE condition with increasing body weight, proteinuria, systolic blood pressure.

The ASA treatment could not attenuate PE condition based on proteinuria score and systolic blood pressure. (A)

Representative data of mean body weight from the groups. (B) Mean proteinuria of the groups. (C) Systolic blood pressure

of the groups.

3 RESULTS

Low-dose of acetylsalicylic acid upregulates

expression of eNOS and down-regulates interleukin-

6 (IL-6) and transforming growth factor-β1 (TGF-

β1).

The expression of IL-6 mRNA was described as

the inflammatory condition of the kidney after PE.

Meanwhile, TGF-β1 expression was represented

chronic effect and pro-fibrotic substance. PE

induction led to significantly higher IL-6 and TGF-

β1 mRNA compared to the Control group. RT-PCR

analysis was demonstrated the beneficial effects of

ASA treatment. ASA treated groups was significantly

lower IL-6 and TGF- β1 mRNA expression compared

to the PE group. PE+ASA125 group was showed the

lowest IL-6 and TGF- β1 mRNA expression. This

group was significantly lower than the PE+ASA75

group.

4 DISCUSSION

This study revealed that a low dose of ASA treatment

did not attenuate blood pressure and proteinuria in the

PE model. However, low doses of ASA treatment had

beneficial effects in inducing eNOS upregulation and

reducing inflammation. ASA125 treatment induced

higher eNOS mRNA expression based on this study.

It seemed that acetylsalicylic acid might increase

eNOS expression in endothelial dysfunction.

Acetylsalicylic acid can stimulate the activation of

eNOS, which can increase NO production for

hypertensive patients in preeclampsia (Suharto et al.,

2014). Nitric oxide can help oxygen transport by

widening the blood vessel wall to facilitate the

transfer of gas from the blood to the tissue. After NOS

synthesizes NO from L-arginine, NO diffuses from

endothelial cells to smooth muscle cells of the blood

vessels and causes an increase in intracellular cyclic

guanosine monophosphate (cGMP). Increased cGMP

will trigger the relaxation of blood vessel muscles to

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

Figure 2. The effect of low-acetyl-salicylic acid on the increased expression of eNOS mRNA and decreased expression of IL-

6 and TGF-β1 mRNA to renal failure in preeclampsia model animals. (A) Representative picture of endothelial function

(eNOS), inflammation marker (IL-6), and fibrosis agent (TGF-β1) expression using RT-PCR. (B) Densitometry analysis of

eNOS mRNA expression from PE kidney model (C) Densitometry analysis of IL-6 mRNA expression from PE kidney model

PE. (D)Densitometry analysis of TGF-β1 mRNA expression from PE kidney model. (E) Representative picture of

glomerulosclerosis based on Periodic Acid-Schiff (PAS) staining in 400× magnification.*:p<0.05 vs control; ##:p<0.01 vs.

PE.***:p<.001 vs PE; ##:p<0.01 vs control.

become a vasodilator to suppress hypertension. Nitric

oxide is known to have properties as an inhibitor of

platelet activation (Jones et al., 2015, Burke et al.,

2016)).

The results showed that preeclampsia's condition

tends to increase the expression of IL-6 and TGF-β1

mRNA, which is often associated with inflammation

and fibrosis. The low dose of ASA treatment in this

study had beneficial effects in lowering IL-6 and

TGF-β1 expression. IL-6 is a pleiotropic cytokine

involved in the regulation of the immune response

and inflammation. IL-6 has biological properties such

as activating transducer signals and activation of

transcription factor STAT3 in renal tubular cells,

stimulation of expression of tissue factor, MCP-1,

matrix degeneration enzyme, and low-density

lipoprotein receptor on macrophage (Jones et al.,

2015). The results of this study can be seen that IL-6

expression tends to increase in preeclamptic

condition compared with the control group. This

condition may be due to preeclampsia, the occurrence

of placental ischemia may contribute to maternal

endothelial cell dysfunction by increasing the

synthesis of IL-6, TNF-α, and IL-8 (Creasy et al.,

2004).

An imbalance between vasoconstrictor and

vasodilator might induce kidney injury with

inflammation and fibrosis. We found that

upregulation of eNOS as vasodilator function in this

study after ASA 125 mg/Kg BW treatment associated

with attenuation of kidney inflammation and fibrotic

marker. This phenomenon might relate to the function

of ASA as anti-inflammatory without influencing

vasodilator capacity in the kidney. A low acetyl-

salicylate acid dose positively affects the balance

between PGI2 as a vasodilator, TXA2 as a

vasoconstrictor, and stimulant platelet aggregation.

At a low dose, acetylsalicylic acid can inhibit TXA2

synthesis without affecting PGI2 synthesis in the

vascular endothelium. According to Villa, research

Low-dose of Acetylsalicylic Acid Upregulates Expression of eNOS mRNA and Downregulates Interleukin-6 (IL-6) and Transforming

Growth Gactor-1 (TGF-1) mRNA in Rat Kidney of Preeclampsia Model

states that a decrease in TXA2 production without a

decrease in PGI2 production can prevent

vasoconstriction and coagulation problems

characteristic of preeclampsia (Villa et al., 2013).

We further examined the effect of low-dose

acetylsalicylic acid administration on the expression

of TGF-β1 as a pro-fibrotic factor in the renal model

of preeclampsia. TGF-β1 is a pro-fibrotic growth

factor that induces renal fibrosis by producing an

extracellular matrix. In this study's results, it can be

seen that TGF-β1 expression tends to increase in

preeclamptic condition compared with the control

group. This phenomenon may occur because, in

preeclampsia conditions, high urinary protein content

may cause pro-inflammatory and pro-fibrotic effects

that contribute to tubulointerstitial damage and loss of

renal function. The main pro-fibrotic factor involved

in this process is TGF-β1 (Kuusniemi et al., 2005).

Increased expression of TGF-β1 may induce renal

fibrosis.

5 CONCLUSION

In conclusion, low doses of ASA treatment might

attenuate inflammation and fibrosis in the kidney

after PE induction. These effects might be associated

with high eNOS mRNA expression.

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JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )